The chromosome is an important target in understanding the cellular effects of ionizing radiation. Of the various components in a chromosome, the DNA molecule is the most critical site for radiation damage. The types and quantities of radiation-induced DNA damage are produced from the sum Of the radicals generated from the direct ionization of the DNA (direct effect) and from reactions of the DNA with free radicals formed in the surrounding environment (indirect effect). It has been suggested that the primary intracellular DNA damaging species produced by radiation is the hydroxyl radical, a bulk water radical. This concept is based largely on studies of DNA irradiated in aqueous systems. However, for cellular DNA, radiation-induced damage can be influenced by other components of the chromosome, e.g. the waters of hydration which comprise a large portion of the water molecules that surround intracellular DNA- Our work suggests that the tightly bound water molecules of the DNA hydration layer contribute to radiation-induced DNA damage primarily via the transfer of charges from these water molecules into the DNA; a mechanism that is different from the induction of DNA damage after irradiation of bulk water (OH.,e(aq-)). Since the mechanism for damaging DNA is different for the hydration water than for the bulk water, interventional strategies that exclusively rely on scavenging hydroxyl radicals to modify DNA damage may have limited success in cells. It is the objective of this proposal to determine the mechanism and extent to which the water of hydration around the DNA contributes to radiation- induced DNA damage. The hypothesis to be tested is that radiation-induced damage in the innermost, tightly bound water molecules of the hydration layer can result in DNA damage that is indistinguishable from that caused by the direct ionization of DNA. First the types and quantities of radiation-induced DNA damage as a function of dose, degree of hydration, DNA conformation, and the presence or absence of oxygen will be examined. This work will then be extended by studying the modifying effects of thiols on the types and quantities of DNA damage that is derived from the tightly bound water molecules in the hydration layer, the direct ionization of the DNA molecule, and the bulk water radicals. Information derived from these studies may be important for understanding the mechanisms by which ionizing radiation causes cancer, mutations and cell lethality, and selecting or designing new radioprotectors that may reduce the deleterious effects of radiation or improve the outcome of radiotherapy.